1. Field of the Invention
The present invention relates to an assay, and kit for carrying out the assay, for determining the physical condition of biological cells in vitro. Particularly, it distinguishes whether cells are alive and proliferating or are dying and, if dying, whether they are in an apoptotic or necrotic condition.
2. Description of the Related Art
Many different cell types are used for the screening of chemical compounds as prospective drugs. Prospective drugs are added to cell cultures in micotitre plates at various concentrations in duplicate or triplicate, so that the average microtitre plate is used to test for many different drugs. After the cells have been incubated for a period of time (depending on the cell type, but usually several days), the effect of the prospective drug on the cells in each well is determined.
The pathway leading to cell death, is important. Cell death may follow two distinct modes: apoptosis and necrosis. Apoptosis is the normal mode and is an active intrinsically controlled process in which the cell commits suicide in response to a wide range of physiological and toxicological signals. It occurs during embryonic development as well as in the adult, controlling immune systems and tissue turnover to regulate a stable balance of tissue mass. Apoptosis also occurs in response to an altered environment, including depletion of growth factors, serum or oxygen and upon exposure to harmful stimuli such as radiation and hyperthermia. Apoptotic cells are removed in vivo by phagocytosis from neighbouring cells and macrophages without invoking an inflammatory response. Necrosis, on the other hand, is a passive pathological event that occurs under severe conditions causing sudden cellular damage and significant loss in tissue architecture. Necrotic cells release their cytoplasmic contents into the extracellular fluid, generating an inflammatory reaction. In in vitro conditions, there is no rapid removal mechanism of apoptotic cells, and so the cells accumulate and ultimately undergo necrotic cell death. Necrotic cell death in these circumstances is termed "secondary necrosis". Ordinary necrosis is termed "primary necrosis".
A determination of the effect of a treatment on the pathway leading to cell death is vital if screening for therapeutic agents is to be successful.
As mentioned above, it is now known that normal cells die by apoptosis, a predetermined pathway which is encoded genetically. Cancer cells have been shown to possess one or more defects which prevent normal cell death from occurring. These defects allow cancer cells to proliferate in an uncontrolled manner.
An assay for determining whether a treatment caused a cancer cell line to undergo cell death by apoptosis is valuable, since it allows one to determine whether or not a treatment can cause the cells to switch back to the normal apoptotic pathway mechanisms of cell death. Such a treatment would of course be preferable to whole scale cytotoxic drugs which may kill normal as well as cancer cells.
In other conditions, such as Alzheimer's Disease, the basis of the condition is cell death. A screening method which can determine whether or not a treatment causes cellular proliferation helps to identify drugs which could correct the cell death exhibited by the condition.
Very potent apoptotic agents may be overlooked because they appear to cause necrosis rather than apoptosis, when in fact they cause apoptosis quickly and the necrosis detected is secondary necrosis which follows apoptosis. It is therefore valuable to detect secondary necrosis.
One of the most significant and early events in necrosis is swelling of mitochondria (hydropic swelling), which is reversible in the early stages providing the causative agent is removed or reversed. However, beyond a certain point the cell will die, and unlike apoptosis, the cells swell, the plasma membrane becomes leaky, resulting in uncontrolled release of the cells constituents such as proteolytic enzymes and inflammatory mediators. In vivo, these substances trigger an inflammatory response in the tissue, resulting in permanent uncontrolled damage, which in certain situations may prove life-threatening.
The feature of a leaky plasma membrane is the basis of a test using propidium iodide (PI) with flow cytometry and allows the viability of the cells to be tested: necrotic cells will stain but apoptotic cells (except in the later stages of apoptosis), or proliferating cells will not. DNA fragmentation in necrosis is not as extensive as in apoptosis and is heterogeneous in size, resulting in a smear on gel electrophorsis. This difference in DNA fragmentation is the basis for the "TUNEL" (Terminal deoxytransferase deoxy Uridine triphosphate-mediated Nick End Labelling) technique which probably represents the best method for determining apoptosis at present and allows differentiation between necrosis and apoptosis. The problem with this method (apart from the very high cost of the equipment) is that it cannot be automated and a set of 20 samples takes up to three hours to prepare, measure and analyse. It is therefore totally unsuitable for screening purposes.
Primary necrosis differs from secondary necrosis in that primary necrosis results in the features detailed above, while necrosis secondary to apoptosis results in increased uptake of the TUNEL label initially, followed by increased uptake of the PI stain due to a leaky plasma membrane in the latter stages of the process.
All of the available methods are limited by the inherent problems of the flow cytometry technology and therefore are not suitable for large scale or high throughput screening.
It is a problem that there are currently no methods available for high throughput assay of the condition of cells, particularly for use in small assay volumes, as in wells of microtitre plates.